Method of large-scale propagation of trees of genus Swietenia

ABSTRACT

Terminal buds or axillary buds of trees of genus Swietenia is cultured on a B5 medium containing 3-indole butyric acid (IBA) and benzylaminopurine (BAP) or its modified medium to induce multiple shoots; the multiple shoots are transplanted to a similar medium containing IBA and BAP to the above and cultured by liquid gyratory culture to efficiently propagate the multiple shoots; then the propagated multiple shoots are transplanted to a similar medium containing IBA and BAP to the above and subjected to liquid stationary culture to efficiently elongate a large number of the shoots; and the elongated shoots are transplanted to a similar medium containing IBA to the above to promote shoot elongation and rooting, thereby producing a large number of young plantlets of trees of genus Swietenia.

TECHNICAL FIELD

The present invention relates to a method for large-scale propagation oftrees of genus Swietenia, particularly Swietenia macrophylla KINGbelonging to the genus Swietenia by tissue culture.

BACKGROUND ART

Trees of genus Swietenia are major afforested trees in tropical zones ofthe world, and their loss due to pest injuries is a serious problem.Recent progress of tissue culture technique has enabled new specieshaving resistances to diseases and pests in many plants, and provisionof trees of genus Swietenia with a resistance to pests is expectable.Generally, conventional techniques such as afforestation based oncuttings, etc. are not acceptable in attempts to propagate useful oldtrees. It is also understood that propagation of elite tree and/or plustrees is hard to conduct.

Recently, several research groups have been studying tissue culturetechniques to solve the problems so far encountered. In this connection,only four successful results have been reported up to now (S.Venketeswaran, M. A. D. L. Dias, F. Sultranbawa, U. V. Weyers (1988):Tissue culture on mahogany tree, Swietenia, Somatic Cell Genetics ofWoody Plants: 147-153; S. K. Lee, A. N. Rao (1988): Plantlet productionof Swietenia macrophylla king through tissue culture, Gard. Bull. Sing.41(1): 11-18; E. Maruyama, K. Ishii, A. Saito, K. Migita (1989):Screening of suitable sterilization of explants and proper media fortissue culture of eleven tree species of Peru-Amazon forest, Journal ofAgricultural Science 33(4): 252-261; and T. Kondo, M. Okamura (1994):Tissue culture of culturing of Woody Plants (Special Issue) '94: 4-5).However these studies are limited to shoot regeneration and induction ofmultiple shoots even when young plantlets germinated under asepticconditions or terminal buds are used as materials, and regeneration ofplantlets is not realized and regeneration and large-scale propagationof young plantlets from mature trees cannot be attained. Thus,development of an efficient culturing method has been keenly desired.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a large-scalepropagation method, which enables large-scale production of trees ofgenus Swietenia, particularly Swietenia macrophylla KING.

As a result of extensive studies on development of a large-scalepropagation method enabling a large-scale production of nursery stocksor mature trees of genus Swietenia, the present inventors succeeded ininduction of multiple shoots having definite buds and/or adventitiousbuds by culturing terminal buds or axillary buds of trees of genusSwietenia and furthermore in efficient propagation of the inducedmultiple shoots. It was further found that efficient induction andpropagation of multiple shoots could be attained particularly by adding3-indole butyric acid (IBA) and benzylaminopurine (BAP) to an inductionmedium for inducing multiple shoots and a medium for propagating themultiple shoots, and furthermore that the propagation efficiency ofmultiple shoots could be increased by liquid gyratory culture forpropagating the multiple shoots. It was also found that efficient shootelongation could be promoted by adding IBA and BAP to the medium and byliquid stationary culture in the elongation of shoots from the multipleshoots. Still furthermore, it was found that further shoot elongationand rooting could be attained by culturing the propagated shoots on amedium containing IBA.

The process for the large-scale propagation of trees of genus Swieteniaaccording to the present invention, which comprises the steps of:

culturing terminal buds or axillary buds of trees of genus Swietenia ona multiple shoots-inducing medium, thereby inducing multiple shootshaving a large number of definite buds and/or adventitious buds;

transplanting the resulting multiple shoots to a propagation medium andpropagating the transplanted multiple shoots;

transplanting the propagated multiple shoots to a shoot elongationmedium and subjecting the transplanted, propagated multiple shoots bystationary culture, thereby promoting shoot elongation; and

transplanting the elongated shoots to a rooting medium, thereby rootingthe transplanted elongated shoots to regenerate the plantlets of treesof genus Swietenia.

BEST MODES FOR CARRYING OUT THE INVENTION

According to the present invention, a large number of young plantlets oftrees of genus Swietenia can be efficiently produced by inducing andpropagating multiple shoots from terminal buds or axillary buds ofnursery stocks and mature trees, then elongating shoots from themultiple shoots so obtained and rooting the elongated shoots.

Trees of genus Swietenia for use in the present invention includes, forexample, Swietenia macrophylla KING.

Culture materials for use in the present invention include, for example,terminal buds or axillary buds excised from nursery stocks and maturetrees. The excised terminal buds or axillary buds are subjected tosurface sterilization with ethanol and sodium hypochlorite or aqueoushydrogen peroxide solution or mercuric chloride (corrosive sublimate)according to the ordinary procedure, and washed with sterilized water anthen cultured on a medium.

The multiple shoots-inducing medium for use in the induction of multipleshoots from terminal buds or axillary buds is an ordinary basal mediumcontaining an inorganic component and a carbon source as essentialcomponents, and also containing a plant growth regulator, a vitamin andamino acid. The inorganic component includes inorganic compoundscontaining such elements as nitrogen, phosphorus, potassium, sodium,calcium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine,cobalt, etc. The carbon source includes carbohydrates such as sucroseand glucose. The plant growth regulator includes, for example, auxin andcytokinin. The auxin includes, for example, 3-indole butyric acid (IBA),naphthalene acetic acid (NAA), etc. The cytokinin includes, for example,benzylaminopurine (BAP), kinetin, zeatin,1-phenyl-3-(1,2,3-Thiadiazol-5-YL)-Urea (Thidiazuron), etc. The vitaminincludes, for example, thiamine, pyridoxine, nicotinic acid, etc. Theamino acid includes, for example, glycine, glutamic acid, lysine, etc.

The medium for use in the actual culture is a medium used for planttissue culture, such as a MS medium (Murashige, T. (1962), Physiol Plant15: 473-497), a B5 medium (Gramborg, O. L. (1968), Exp. Cell Res. 50:151-158), a WP medium (Lloyd, G. (1981), Int. Plant prop. Soc. 30:421-427), an BTM medium (Chalupa, V. (1984) Biologia Plnt. 26: 374-377),etc. Or, their modified media such as a modified MS medium, a modifiedB5 medium, a modified WP medium, a modified BTM medium, etc. may beused, where the modified media designate media containing each ofcomponents in the basic media such as MS, B5, WP and BTM media at achanged concentration of, for example, half or one-fourth of theconcentration in the basic media.

Particularly preferable are a B5 medium and its modified medium. Topromote induction and growth of definite buds and adventitious buds, itis preferable to use a medium containing 0.02 to 1.0 mg/l of BAP or notmore than 0.02 mg/l of IBA together with 0.02 to 1.0 mg/l of BAP.

Then, the multiple shoots so obtained are transplanted to a propagationmedium to efficiently propagate the transplanted multiple shoots.

As a propagation medium, the above-mentioned basal medium can be used.Particularly, a medium containing an inorganic component, a carbonsource, a vitamin, amino acid, etc. and also containing 0.02 to 0.2 mg/lof IBA and 0.2 to 2.0 mg/l of BAP is preferable. In actual culture, itis preferable to use a B5 medium containing such amounts of IBA and BAPas above or its modified medium. A solid or liquid medium is preferablyused for the culture, and particularly a liquid gyratory culture ispreferably used. More specifically a liquid gyratory culture under agyratory condition of 60 to 100 rpm, preferably 70 to 80 rpm, ispreferable.

Then, the propagated multiple shoots are translated to a shootelongation medium to efficiently elongate shoots from the transplantedmultiple shoots.

As a shoot elongation medium, the abovementioned basal medium can beused. Particularly, a medium containing an inorganic component, a carbonsource, a vitamin, amino acid, etc. and also containing 0.02 to 0.2 mg/lof IBA and 0.2 to 2.0 mg/l of BAP is preferable. More specifically, a B5medium containing such amounts of IBA and BAP as above or its modifiedmedium is preferably used. A solid or liquid medium is preferably usedfor the culture, and particularly a liquid stationary culture ispreferable.

Then, the elongated shoots are transplanted to a rooting medium with orwithout cutting to single shoots to further elongate and root theshoots.

As a rooting medium, the above-mentioned basal medium can be used, andparticularly a medium containing an inorganic component, a carbonsource, a vitamin, amino acid, etc. and also containing 1.0 to 5.0 mg/lof IBA is preferable. Actually, a B5 medium containing such an amount ofIBA as above or its modified medium is preferable.

According to the present invention it is preferable to culture elongatedshoots on a modified MS medium containing 1.0 to 3.0 mg/l of IBA, andtransplant and culture the further elongated shoots to and on a modifiedB5 medium containing 1.0 to 3.0 mg/l of IBA, thereby rooting the shootsand regenerating the plantlets. The modified MS medium and modified B5medium to be used each preferably contain 2.5 mg/l of IBA. Culturingtime on the modified MS medium and modified B5 medium depends onculturing conditions, and usually is 15 days to one month, andparticularly preferably about one month. Preferable culturing conditionsare a temperature of 23° to 30° C., a humidity of 60 to 100%, and alight intensity of 3,000 to 9,000 Lux.

According to the present invention, it is preferable, after culturing onthe above-mentioned modified MS medium and modified B5 medium to culturethe shoots on a modified MS medium containing 1.0 to 3.0 mg/l or IBA for15 days to one month, preferably for about one month. It is particularlypreferable to use a modified MS medium containing 2.5 mg/l of IBA.Culturing conditions on the modified MS medium are the same as above.

By culturing in the above-mentioned specific modified medium, the shootrooting rate can be elevated, whereas the withering rate can be lowered.

According to one of preferable modes for carrying out the present methodfor the large-scale propagation of trees of genus Swietenia by tissueculture, terminal buds or axillary buds of trees of genus Swietenia isat first cultured on a B5 medium containing BAP or IBA together with BAPor its modified medium to induce multiple shoots therefrom; then themultiple shoots are transplanted to a propagation medium containing IBAand BAP and cultured by a liquid gyratory culture to propagate themultiple shoots; then the propagated multiple shoots are transplanted toa shoot elongation medium containing IBA and BAP and cultured by liquidstationary culture, thereby promoting to elongate the shoots; and thenthe elongated shoots are transplanted to a rooting medium containing IBAand cultured, thereby producing young plantlets of trees of genusSwietenia.

EXAMPLE

The present invention will be described in detail below, referring toExample.

Nursery stocks of Swietenia macrophylla obtained from Indonesia (0.2 to2.0 m high) were made to grow in a greenhouse to heights of 1.0 to 6.0m, and then materials were excised therefrom.

(1) Sterilization of Materials

Terminal buds were excised from mature trees of Swietenia macrophyllaand subjected to surface sterilization in 70% ethanol for 30 seconds andthen in 2% sodium hypochlorite for 6 minutes, washed several times withsterilized water and dried on a sterilized filter paper.

(2) Induction of Multiple Shoots

To a modified B5 medium (which was a B5 medium amounts of all thecomponents were each made a half were added 30 g/l of sucrose, and 0 to0.02 mg/l of IBA and 0.02 to 1.0 mg/l of BAP as plant growth regulators,followed by pH adjustment to 5.7 and sterilization. The medium soprepared was used as a multiple shoots-inducing medium, and was used ata culturing temperature of 26±2° C. under illumination of fluorescentlamps for 16 hours per day (3,000 to 9,000 Lux). One to 3 months afterthe start of culturing, multiple shoots were induced.

Relations between the amounts of IBA and BAP added shoots the medium andnumber of formed multiple shoots are shown in Table 1.

TABLE 1 Conditions for inducing multiple shoots Number of formedmultiple shoots/Number Plant growth regulator of total tested terminalIBA (mg/l) BAP (mg/l) buds 0 0.02 1/20 0 0.2 5/20 0 1.0 8/20 0.02 0.020/20 0.02 0.2 7/20 0.02 1.0 15/20 

As is evident from the results shown in Table 1, addition of 0.02 mg/lof IBA and 1.0 mg/l of BAP is particularly effective.

(3) Propagation of Multiple Shoots

Multiple shoots obtained in (2) were cut to single shoots, which weretransplanted to a propagation medium. A modified B5 medium (which wasthe same as used in (2) containing 0.02 to 0.2 mg/l of IBA and 0.2 to2.0 mg/l of BAP was used as a propagation medium after pH adjustment to5.7 and sterilization. Culturing temperature was 26±2° C. underillumination of fluorescent lamps for 16 hours per day (3.00 to 9.000Lux). As a result, about 6-fold shoots could be obtained 1 to 2 monthsafter the start of culturing.

Relations between the amounts of IBA and BAP added to the medium andnumber of propagated shoots are shown in Table 2.

TABLE 2 Conditions for propagating multiple shoots Average number ofPlant growth regulator propagated shoots/ IBA BAP Multiple shoot 0.020.2 4.1 0.02 0.5 4.7 0.02 2.0 1.2 0.2 0.2 5.9 0.2 0.5 3.1 0.2 2.0 0.7

As is evident from the results shown in Table 2, addition of 0.2 mg/l ofIBA and 0.2 mg/l of BAP is particularly effective.

(4) Shoot Elongation

Multiple shoots propagated in (3) were transplanted as such to a shootelongation medium. A modified B5 medium (which was the same as used in(2)) containing 0.02 mg/l of IBA and 0.2 to 2.0 mg/l of BAP was used asa shoot elongation medium after pH adjustment to 5.7 and sterilization.Culturing temperature was 26±2° C. under illumination of fluorescentlamps for 16 hours per day (3,000 to 9,000 Lux). As a result, all theshoots could be elongated to lengths of 5 to 10 mm one month after thestart of culturing, and addition of 0.2 mg/l of IBA and 0.2 mg/l of BAPis particularly effective.

(5) Rooting

A large number of shoots obtained in (4) are cut to single shoots, whichwere then transplanted to a rooting medium. A modified B5 medium (whichwas the same as in (2)) containing 1.0 to 5.0 mg/l of IBA and 2.8 g/l ofgerlite was used as a rotting medium after pH adjustment to 5.7 andsterilization. Culturing temperature was 26±2° C. under illumination offluorescent lamps for 16 hours per day (3,000 to 9,000 Lux). It wasobserved that 18.5% of the single shoots were rooted one to two monthsafter the start of culturing with shoot elongation of 3 to 5 cm andformation of 2 to 5 leaves.

Relations between the amount of IBA added to the medium and number ofrooted single shoots are shown in Table 3.

TABLE 3 Rooting conditions Plant growth regulator Number of rooted IBA(mg/l) single shoots 1.0 1/27 2.0 2/30 2.5 5/27 3.0 0/26 4.0 1/25 5.00/26 10.0 0/26

As is evident from Tale 3, addition of 2.5 mg/l of IBA is effective.

(6) Rooting

A large number of shoots obtained in (4) were cut to single shoots whichwere cultured in the following procedures:

{circle around (1)} The single shoots were cultured on a modified B5medium containing 0 to 10.0 mg/l of IBA for 3 months, whiletransplanting the shoots to a fresh medium of the same composition everymonth.

{circle around (2)} The single shoots were cultured on a modified MSmedium containing 0 to 10.0 mg/l of IBA for 3 months, whiletransplanting the shoots to a fresh medium of the same composition everymonth.

{circle around (3)} The single shoots were cultured on a modified MSmedium containing 0 to 10.0 mg/l of IBA for one month, then on amodified B5 medium containing 0 to 10.0 mg/l of IBA for one month andfurther on a modified MS medium containing 0 to 10.0 mg/l of IBA for onemonth.

{circle around (4)} The single shoots were cultured on a modified B5medium containing 0 to 10.0 mg/l of IBA for one month, then on amodified MS medium containing 0 to 10.0 mg/l of IBA for one month andfurther on a modified B5 medium containing 0 to 10.0 mg/l of IBA for onemonth.

All the above {circle around (1)}-{circle around (4)} cultures wereconducted under conditions wherein a temperature was 26±2° C., ahumidity was 60 to 90% and a light intensity was 3,000 to 9,000 Lux.Further, all the modified media in the above {circle around (1)}-{circlearound (4)} contained 2.8 g/l of gelright.

Results of culturing under the conditions of {circle around (1)} to{circle around (4)} (rooting rate and withering rate) are shown inTables 4 to 7.

TABLE 4 Rooting condition {circle around (1)} Medium Rooting rate (%)Withering rate (%) For For For For For For For For For first secondthird first second third first second third one one one IBA one one oneone one one month month month (mg/l) month month month month month monthModified Modified Modified 0 0.0% 0.0% 0.0% 92.3% 100.0% 100.0% B5 B5 B50.1 0.0% 0.0% 0.0% 84.6% 100.0% 100.0% 1.0 0.0% 3.7% 3.7% 14.8% 55.6%96.3% 2.0 0.0% 6.7% 6.7% 13.3% 40.0% 73.3% 2.5 0.0% 18.5% 18.5% 11.1%40.7% 66.7% 3.0 0.0% 0.0% 0.0% 26.9% 57.7% 73.1% 4.0 0.0% 4.0% 4.0%40.0% 72.0% 100.0% 5.0 0.0% 0.0% 0.0% 53.8% 84.6% 100.0% 10.0 0.0% 0.0%0.0% 80.8% 96.2% 100.0%

TABLE 5 Rooting conditions {circle around (2)} Medium Rooting rate (%)Withering rate (%) For For For For For For For For For first secondthird first second third first second third one one one IBA one one oneone one one month month month (mg/l) month month month month month monthModified Modified Modified 0 0.0% 0.0% 0.0% 19.2% 84.6% 92.3% MS MS MS0.1 0.0% 0.0% 0.0% 0.0% 69.2% 84.6% 1.0 0.0% 3.8% 3.8% 0.0% 46.2% 73.1%2.0 0.0% 3.8% 11.5% 0.0% 30.8% 76.9% 2.5 0.0% 13.3% 16.7% 0.0% 26.7%66.7% 3.0 0.0% 3.7% 11.1% 0.0% 55.6% 70.4% 4.0 0.0% 0.0% 0.0% 10.3%58.6% 96.6% 5.0 0.0% 0.0% 0.0% 46.2% 76.9% 100.0% 10.0 0.0% 0.0% 0.0%65.4% 80.8% 100.0%

TABLE 6 Rooting conditions {circle around (3)} Medium Rooting rate (%)Withering rate (%) For For For For For For For For For first secondthird first second third first second third one one one IBA one one oneone one one month month month (mg/l) month month month month month monthModified Modified Modified 0 0.0% 0.0% 0.0% 26.9% 69.2% 76.9% MS MS MS0.1 0.0% 0.0% 0.0% 7.7% 73.1% 84.6% 1.0 0.0% 10.0% 40.0% 0.0% 30.0%50.0% 2.0 0.0% 10.0% 40.0% 0.0% 30.0% 40.0% 2.5 0.0% 40.0% 70.0% 0.0%0.0% 20.0% 3.0 0.0% 10.0% 30.0% 0.0% 50.0% 50.0% 4.0 0.0% 0.0% 0.0%20.0% 60.0% 80.0% 5.0 0.0% 0.0% 0.0% 40.0% 50.0% 100.0% 10.0 0.0% 0.0%0.0% 50.0% 100.0% 100.0%

TABLE 7 Rooting conditions {circle around (4)} Medium Rooting rate (%)Withering rate (%) For For For For For For For For For first secondthird first second third first second third one one one IBA one one oneone one one month month month (mg/l) month month month month month monthModified Modified Modified 0 0.0% 0.0% 0.0% 84.6% 92.3% 100.0% MS MS MS0.1 0.0% 0.0% 0.0% 84.6% 88.5% 100.0% 1.0 0.0% 0.0% 10.0% 10.0% 50.0%70.0% 2.0 0.0% 10.0% 10.0% 0.0% 40.0% 50.0% 2.5 0.0% 30.0% 50.0% 0.0%40.0% 40.0% 3.0 0.0% 10.0% 20.0% 0.0% 60.0% 70.0% 4.0 0.0% 0.0% 0.0%40.0% 70.0% 100.0% 5.0 0.0% 0.0% 0.0% 50.0% 90.0% 100.0% 10.0 0.0% 0.0%0.0% 70.0% 100.0% 100.0%

As is evident from the results shown in Tables 4 to 7, it was observedthat in case of culturing according the procedure {circle around (3)},70.0% of the single shoots were rooted 3 months after the start ofculturing with shoot elongation of 3 to 10 cm and formation of 2 to 5new leaves, and addition of 2.5 mg/l of IBA is particularly effective.

That is, it was found more effective to culture the shoots on a modifiedMS medium containing 2.5 mg/l of IBA for one month, then on a modifiedB5 medium containing 2.5 mg/l of IBA for one month and further on amodified MS medium containing 2.5 mg/l of IBA for one month than tocontinuously culture them on a single kind of medium.

INDUSTRIAL UTILITY

According to the present invention, a large number of aseptic youngplantlets for tissue culture of trees of genus Swietenia and nurserystocks and cuttings for cuttage can be produced in vitro for a shorttime with a high possibility of large-scale production of nursery stocksof trees of genus Swietenia.

What is claimed is:
 1. A method for large-scale production of trees ofgenus Swietenia, which comprises the steps of: culturing terminal budsor axillary buds of trees of genus Swietenia on a multipleshoots-inducing medium wherein a B5 medium containing 0.02 to 1.0 mg/lof benzylaminopurine (BAP), or a B5 medium containing; not more than0.02 mg/l of 3-indole butyric acid (IBA) together with 0.02 to 1.0 mg/lof BAP, or its modified B5 medium is used as the multipleshoots-inducing medium for inducting multiple shoots, thereby inducingmultiple shoots having a large number of definite buds and/oradventitions buds; transplanting the multiple shoots to a propagationmedium, thereby propagating the multiple shoots; transplanting thepropagated multiple shoots to a shoot elongation medium and subjectingthe shoots to stationary culture, wherein the elongate shoots arecultured on a modified MS medium containing 1.0 to 3.0 mg/l of IBA,thereby promoting shoot elongation; and transplanting the elongatedshoots to a modified B5 medium containing 1.0 to 3.0 mg/l of IBA andcultured, and rooting the shoots, thereby regenerating plantlets oftrees of genus Swietenia.
 2. A method according to claim 1, wherein themultiple shoots are propagated on the propagation medium by liquidgyratory culture, thereby increasing a propagation rate of multipleshoots.
 3. A method according to claim 2, wherein a B5 medium containing0.02 to 0.2 mg/l of IBA and 0.2 to 2.0 mg/l of BAP or its modifiedmedium is used as the propagation medium.
 4. A method according to claim1, wherein a B5 medium containing 0.02 to 0.2 mg/l of IBA and 0.2 to 2.0mg/l of BAP or its modified medium is used as the propagation medium. 5.A method according to claim 1, wherein the shoot elongation of themultiple shoots on the shoot elongation medium is carried out by liquidstationary culture.
 6. A method according to claim 1, wherein a B5medium containing 0.02 to 0.2 mg/l of IBA and 0.2 to 2.0 mg/l of BAP orits modified medium is used as the shoot elongation medium.
 7. A methodaccording to claim 1, wherein a B5 medium containing 1.0 to 5.0 mg/l ofIBA or its modified medium is used a the rotting medium.
 8. A methodaccording to claim 1, wherein the trees of genus Swietenia is Swieteniamacrophylla King.
 9. A method according to claim 1, wherein a modifiedMS medium containing 2.5 mg/l of IBA and a modified B5 medium containing2.5 mg/l of IBA are used as the modified MS medium and the modified B5medium, respectively.
 10. A method according to claim 9, wherein cultureis carried out on the modified MS medium for about one month and then onthe modified B5 medium for about one month.
 11. A method according toclaim 9, wherein culturing is further carried out on a modified MSmedium containing 1.0 to 3.0 mg/l of IBA.
 12. A method according toclaim 1, wherein culture is carried out on the modified MS medium forabout one month and then on the modified B5 medium for about one month.13. A method according to any one of claim 1, wherein culturing isfurther carried out on a modified MS medium containing 1.0 to 3.0 mg/iof IBA.
 14. A method according to claim 13, wherein a modified MS mediumcontaining 2.5 mg/l of IBA is used as the modified MS medium.
 15. Amethod according to claim 14, wherein culturing is carried out on themodified MS medium for about one month.
 16. A method according to claim13, wherein culturing is carried out on the modified MS medium for aboutone month.
 17. A method according to claim 1, wherein B5 mediumcontaining 0.02 to 0.2 mg/l of IBA and 0.2 to 2.0 mg/l of BAP or itsmodified medium is used as the propagation medium.